Biological production of proteins through recombinant DNA technology has been one of the leading aspects in biotechnology research over the last decade. To achieve economically viable levels of expression while still obtaining a biologically active protein, both eukaryotic and prokaryotic systems have been studied. Prokaryotic systems typically utilize Escherichia coli as the bacterial host of choice for the expression of heterologous genes for both practical and economical considerations. However, the insertion of cloned DNA sequences into an expression unit does not guarantee efficient gene expression when the expression unit is introduced into the bacterial host cell.
Hence, despite the versatility and efficacy of its expression vectors, with levels of expression in the range of 20 to 50% of total cellular proteins, E. coli suffers several limitations for the expression of different categories of heterologous proteins especially those that undergo complex post-translational modifications such as many viral and mammalian proteins. Those limitations include inappropriate or lack of post-translational modifications, incorrect folding, proteolytic degradation, inefficient secretion and, recently reported, amino acid misincorporation.
Because of the limitations described above for E. coli expression systems, efforts have been directed towards the development of more sophisticated expression systems including other prokaryotes, lower eukaryotes such as yeast, and higher eucaryotes such as mammalian and insect cells. From the review of the vast literature reports on the expression of recombinant proteins appears to emerge the increasingly accepted notion that there is no "universal expression system". The current trends in the field is to tailor the development of expression systems to fit the specific expression needs. It is in that perspective that insect virus vectors and adenovirus vectors have been initially developed, mainly to exploit their respective capacity to express recombinant proteins in insect and human cells.
Poxvirus research, and more particularly the use of vaccinia virus, a prototypic member of the group of poxviruses, has led to eukaryotic cloning and expression of vectors useful in various biological and medical applications. In 1982, Panicali and Paoletti reported that endogenous subgenomic elements could be inserted into infectious progeny vaccinia virus via recombination in vivo (1982, Proc. Natl. Acad. Sci. USA, 1979: 4927-4931); and subsequently demonstrated that they could insert foreign genes into infectious vaccinia virus and obtain pure cultures of recombinant vaccinia virus expressing the foreign gene.
It was reported that vaccinia virus appear to have several advantages over other eukaryotic vectors. Most noteworthy was the fact that virus infectivity was not impaired by insertion and expression of foreign gene in contrast to defective SV40 and retrovirus vectors. Although, vaccinia virus has been successfully used as an expression vector through the insertion of foreign genes into a non-essential region of the viral genome via homologous recombination, some drawbacks have also been associated with the use of this virus. The most difficult problem appears to reside in the fact that vaccinia expression vectors are not capable of producing abundant foreign proteins because of the absence of known strong promoters.
Baculovirus vectors have also been used for the expression of foreign genes in insect cells. Indeed, in the case of baculovirus, two very strong and very late promoters are responsible for the expression of two extremely abundant proteins, polyhedrin and p10, which can together constitute as much as 50% of total cellular proteins in baculovirus-infected cells. Autographa californica nucleopolyhedrosis virus (AcNPV) is the prototype virus of the family Baculoviridae. This virus has a wide host range and infects a large number of species of lepidoptera insects.
AcNPV possesses several properties that make this virus ideally suited as an expression vector for cloned eukaryotic genes. Since occlusion of the virus is not absolutely essential for viral growth, the polyhedrin gene provides a non-essential region of the AcNPV genome in which foreign DNA may be inserted. Placing foreign genes of interest under the control of either the polyhedrin or the p10 promoter have led in the best cases to production of recombinant proteins at 20-25% of total cellular proteins. The rapid construction of efficient transfer vectors has also been facilitated by the relatively low complexity of gene regulation in the expression of the polyhedrin and p10 baculovirus genes.
Using the properties of AcNPV, a wide variety of eukaryotic and prokaryotic genes have been expressed successfully with baculovirus vectors in insect cells. However, expression levels for different genes inserted into the same vector are often different and are related to the length and nature of the leader sequence preceding the foreign gene. Even in the best available vectors, there is some variability in expression levels depending on factors such as the nature of the gene and the protein expressed. Furthermore, careful characterization of numerous recombinant proteins has pointed to some problems in post-translational modifications in insect cells, such as impaired glycosylation, incomplete proteolytic cleavage of polyprotein precursors, and inefficient secretion. This would appear to preclude the utilization of this expression system for the production of numerous complex mammalian proteins. In this regard, other alternatives better suited for the expression of mammalian proteins, such as adenovirus vectors, are being investigated.
Adenoviruses (Ad) have first been isolated over three decades ago. Since then, many efforts have been invested into defining their biological properties. The intimate association that these viruses have with their host during infection has potentiated their value as tools for exploring the mechanisms of macromoleculer biosynthesis in mammalian cells. The temporal pattern of adenovirus infection of human cells is generally demarcated by two phases of expression, early and late, which are separated by the onset of replication after about 8 hours of infcetion. Early in infection, at least 7 promoters are active, generating transcripts from early regions 1-4. Over 30 messages corresponding to the early regions have been identified by RNA analysis and/or cDNA cloning.
In contrast, the high levels of expression of the abundant viral late proteins are the result of the strong transcriptional activity of one promoter, the major late promoter (MLP) which is responsible for the production of some twenty late proteins encoded by an equivalent number of mRNAs. These mRNAs are all derived from one very long primary transcript by maturation processes involving differential splicing and polyadenylation events. Among those late proteins, three structural proteins, namely hexon (15-20% of total cellular proteins), fiber (8-10%), and penton (2-4%), and one non-structural protein named 100K (5-10%), constitute collectively as much as 35% of total cellular proteins in Ad-infected cells, whereas the remaining minor late proteins would constitute some 5%. FIG. 1 shows an autoradiogram of the late structural proteins metabolically labelled with .sup.35 S methionine from adenovirus infected 293 cells. The AdPyR39 recombinant was produced following the description provided by Massie et al. (1986, Mol. Cell. Biology, 6:2872-2883, hereby incorporated by reference). The relative abundance of these late viral proteins can fluctuate depending on infection conditions. However, little is known about the mechanism which regulates this phenomenon. In any case, only a small portion of the structural proteins which are synthesized in copious amount, 20-30% of the hexon and 1-5% of penton and fiber respectively, are assembled into functional nucleocapsids. Therefore, it was soon realized that appropriate manipulations of Ad genome could potentially result in the construction of Ad recombinants expressing foreign proteins at very high levels.
The first human adenovirus (Ad) vectors have been developed in the early 1980's. These vectors have been used to express a wide variety of viral and cellular genes (for a complete review, see Berkler, 1988, Biotechniques 6:616-629, hereby incorporated by reference). Currently, there are three potential commercial applications for Ad vectors, namely in 1) high level expression of heterologous proteins, 2) live viral sub-unit vaccines and 3) gene transfer vectors for establishing stable cell lines or gene therapy.
Adenovirus vectors appeared promising for expression of high levels of protein, since transcription from the major late promoter was so efficient and high levels of translation were accompanied by host protein synthesis shut-off late in infection, facilitating protein isolation. Furthermore, human adenoviruses can replicate efficiently to very high titers (10.sup.9 -10.sup.10 pfu/ml) in human cells, as well as other mammalian cells; and adenoviruses produce their late proteins at levels than reach 30 to 40% of total cellular proteins. Finally, they can be propagated in suspension cultures thereby demonstrating a clear potential for large scale production.
The construction of helper-free Ad recombinants expressing foreign proteins at very high levels is generally accomplished by using MLP-based expression cassettes inserted in the deleted E1 region. The ectopic MLP sequences found in those expression cassettes have incorporated between 200 and 700 bp upstream, and 33 bp downstream of the transcriptional start site. Those sequences were expected to include all of the required elements that confer full transcriptional activity to the endogenous MLP. However, the majority of recombinant adenoviruses constructed thus far express only low to moderate levels of heterologous proteins. These levels are usually lower than the normal levels of adenovirus late proteins. Only a hand-full of examples of Ad recombinants were shown to express recombinant proteins at levels that rival the amount of some of the abundant adenoviral late proteins. Examples include AdSVR112 (Gluzman et al., 1982, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory Press, N.Y., pp. 187-192) which expresses the SV40 large T antigen at 3-4% of total cellular proteins; and Ad5-RR2.sup.HSV which expresses the HSV ribonucleotide reductase subunit 2 (R2) at 4-5% of total cellular proteins (Lamarche et al., 1990, J. Gen. Virology, 71:1785-1792, hereby incorporated by reference).
Also described in the literature are adenovirus recombinants that appear to produce foreign proteins at levels which are somewhat between the level at which the 100 K protein is produced and the level at which the fiber protein is produced, although no accurate quantitation was reported in those latter cases. Thus, it seems that none of these Ad recombinants succeeded in expressing their heterologous protein at a level equivalent to the level of hexon or fiber which are respectively the first and second most abundant proteins in Ad-infected cells. These attempts indicate that because of the complexity in the regulation of gene expression in adenoviruses, their full potential as high level expression vectors has yet to be realized.
A better understanding of the molecular mechanisms underlying the complex regulation of gene expression in adenoviruses is essential in order to construct transfer vectors which exploit the full potential for high level expression in this system. One example among the best recombinant cistron assembled so far for high level expression of foreign genes in adenovirus is represented in the transfer vector pAdBM1 (Lamarche et al., supra). In pAdBM1, the expression cassette includes sequentially: the strong major late promoter (MLP), a high efficiency translational leader (Ad2 tripartite leader), splicing signals, a cloning site, and multiple polyadenylation sites. Given that in Ad recombinants derived from pAdBM1 the ectopic MLP drives the expression of only one mRNA, whereas the endogenous MLP produces more than 20 mRNAs, one would have expected that the level of expression from the ectopic MLP would be equivalent to or approaching the sum of the late viral proteins. Since this has not been observed, it appeared reasonable to conclude that the MLP functions much less efficiently when taken out of its native environment. Whether a promoter structure similar to, or mimicking that of, the native MLP configuration could be obtained was not clear as the number of combinations in which a promoter could be linked to enhancers and other transcriptional and translational control elements is essentially infinite despite the finding by Leong et al. (1990, J. Virology, 64: 51-60) that sequences mapping between +30 and +130 have been shown to play a critical role in the induction of sequence-specific binding proteins by transcription of the Ad MLP during the late phase of infection.
The present invention relates to the development of an improved Ad expression system utilizing an improved and novel Ad expression vector termed pAdBM5. The construction of pAdBM5 included the introduction of enhancer sequences to further stimulate the transcriptional activity of the ectopic MLP.
Traditional enhancers are expression elements that have been described as having the following properties: 1) containing repeated sequences that can function independently; 2) acting over distances, sometimes considerable (even up to thousands of bases); 3) may function in either orientation; 4) may function in a position independent manner, and can be within or downstream of the transcribed region, but can only function in cis (if several promoters lie nearby, the enhancer may preferentially act on the closest); 5) may function in a cell type or tissue-specific manner (Kriegler, 1990, Methods in Enzymology, 185: 512-527). Studies, however, have shown that the properties of enhancers are even more highly varied and can function in a variety of ways. For example, the effects of the variation of position of the SV40 enhancer on the expression of multiple transcription units in a single plasmid revealed two types of position effects. One position effect is called promoter occlusion and results in reduced transcription at a downstream promoter if transcription is initiated at a nearby upstream promoter. This effect does not involve enhancer elements directly, even though the effect is most pronounced when the downstream promoter lacks an enhancer element. The second effect stems from the ability of promoter sequences to reduce the effect of a single enhancer element on other promoters in the same plasmid. Thus, according to Kriegler, the SV40 enhancer element is a complex structure whose function is subject to some position effects and whose cell-type-specific activation is dependent, in part, on the absence or presence of active cellular factors or proximal sequences.
Another example given by Kriegler is one of a viral enhancer described in the hepatitis B virus. This enhancer is located 3' to the hepatitis B virus surface antigen coding sequences but is contained within the mature viral transcripts. Authors have reported that the HBV enhancer can dramatically increase expression levels of genes controlled by the SV40 enhancer/promoter but only when the enhancer is located within the transcribed region of the gene. Further, this effect appears to be orientation dependent, a violation of already unsettled enhancer rules.
Thus, from previous studies, it would not be obvious to one skilled in the art how to use enhancers to increase transcription from the Ad MLP; i.e. it would have been difficult to predict what enhancer sequences may be used, or where or how it may be placed in the genome of a recombinant adenovirus vector to enhance expression levels.
Recently, it has been shown that a number of cis-acting sequences are essential to confer full transcriptional activity of the MLP during the late phase of infection (Mondesart et al., 1991, Nucleic Acids Research 19:3221-3228, hereby incorporated by reference). These include an upstream element (UE) between -67 and -49 relative to the transcriptional start site; a TATA box centered at -28; and an initiator element encompassing the transcription start site. In addition, some downstream elements (DE) have been mapped and designated, RI (+37 to +68), DE1 (+85 to +96) or R2 (+80 to +105), and DE2 (+109 to +124) or R3 (+105 to +125) (see FIG. 2). While the UE, the TATA box and the R1 downstream element have been shown to be important for basal transcriptional activity of the MLP both at early and late times, the DE (DE1 and DE2) would be essential for late phase specific activation. DE1 and DE2 are functionally redundant and probably bind to the same transcription factor(s). They may also interact synergistically with the UE by an unknown mechanism, to bring about their late phase specific transcriptional activation. At this point, it is not clear whether these cis-acting sequences are "enhancer-like" or downstream promoter elements and whether enhanced expression could be obtained by inserting them in a transfer vector. These sequences are missing in all of the MLP currently used in Ad transfer vectors described so far. The inherent difficulty in properly evaluating the position at which enhancer-like or downstream promoter elements could be inserted to enhance expression still remains to be solved.